UNIPROT:P10415 - FACTA Search

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Query: UNIPROT:P10415 (Bcl-2)
33,771 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Endogenously generated or exogenously supplied nitric oxide (NO)-induced apoptotic cell death in the mouse macrophage cell line RAW 264.7. Apoptotic signaling caused an early accumulation of the tumor suppressor p53 prior to DNA fragmentation. Contrary to the notion of specific activating signals, inhibitory transduction mechanisms largely remain unknown. Therefore, RAW 264.7 macrophages were stably transfected with human Bcl-2, an anti-apoptotic protein. Bcl-2 transfectants showed substantial protection from cell death induced following the exposure to NO donors such as S-nitrosoglutathione (GSNO) and spermine-NO. In contrast, in RAW 264. 7 parent or in neomycin control-transformed cells, these NO donors induced internucleosomal DNA cleavage in a dose-dependent manner. Similarly, expression of the inducible NO synthase in response to lipopolysaccharide and interferon-gamma also caused apoptosis in RAW macrophages and neo controls within 24 h. In contrast, Bcl-2 transfectants appeared highly resistant, although inducible NO synthase levels increased along with concomitant nitrite production similar to control cells. The expression of p53 and Bax was also explored in controls and Bcl-2 transfectants after GSNO addition. GSNO induced p53 expression in Bcl-2 transfectants at levels comparable with nontransfected RAW macrophages. Moreover, GSNO induced increases in the steady-state levels of Bax protein in parental and Bcl-2-transfected cells. We conclude therefore, that Bcl-2 acts downstream of p53, presumably nullifying the NO-mediated increase in Bax protein in RAW 264.7 cells.
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PMID:Bcl-2 protects macrophages from nitric oxide-induced apoptosis. 870 45

Polymorphonuclear leukocytes (PMN) isolated from the oral cavity of healthy human volunteers, spontaneously generated superoxide, nitric oxide (NO) and other reactive oxygen species (ROS) which exhibited strong luminol chemiluminescence (LCL). To understand the physiological roles of oral PMN (OPMN), biochemical properties of the cells were analyzed. Biochemical analysis revealed that OPMN were already primed under physiological conditions. Western blot analysis revealed that they strongly expressed the inducible type of NO synthase (NOS II) and exhibited the activity to catalyze tyrosine phosphorylation of various proteins including a 115 kDa protein (cbl product). OPMN also generated H2O2 and .OH by some superoxide dismutase (SOD)-sensitive mechanism and released myeloperoxidase (MPO). Kinetic analysis using specific inhibitors revealed that OCl- generated by OPMN was predominantly responsible for the enhanced LCL. During the incubation under standard culture conditions, OPMN underwent apoptosis which proceeded more rapidly than that of the circulating PMN (CPMN). Immunochemical analysis revealed that expression of apoptosis-related gene products, such as Bcl-2, Bcl-xL and Bax, was below detectable levels with both cell types. However, caspase-3 but not caspase-1 was markedly activated in OPMN. These results indicate that the primed OPMN spontaneously generate ROS and play an important role in the defense mechanism in the oral cavity and that the generated ROS activate caspase-3 thereby inducing apoptosis of the cells.
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PMID:Biochemical properties of human oral polymorphonuclear leukocytes. 970 29

Our group recently reported that cultured sheep pulmonary artery endothelial cells (SPAECs) became resistant to lipopolysaccharide (LPS)-induced apoptosis several days after constitutive synthesis of nitric oxide (NO) after adenoviral (Ad) transfer of inducible NO synthase (iNOS) or exposure to the NO donor S-nitroso-N-acetylpenicillamine (SNAP) (E. Tzeng, Y.-M. Kim, B. R. Pitt, A. Lizonova, I. Kovesdi, and T. R. Billiar. Surgery 122: 255-263, 1997). In the present study, we confirmed this observation by establishing stable transfectants after retroviral gene transfer [replication-deficient retrovirus (DFG)] of human iNOS (DFG-iNOS) SPAECs and then used all three approaches (Ad, DFG, and SNAP) to determine underlying mechanisms of this phenomenon. Continuous endogenous production of NO in itself did not cause apoptosis as assessed by phase-contrast microscopy, nuclear morphology, and internucleosomal DNA fragmentation. Prolonged (72-96 h) synthesis of NO, however, after DFG- or replication-deficient adenovirus (Ad. CMV)-iNOS or SNAP (100 microM, 96 h) inhibited LPS-induced apoptosis. The kinetics of such protection suggested that NO may be inducing other gene products. Ad-mediated transfer of manganese superoxide dismutase (MnSOD) decreased the sensitivity of wild-type SPAECs to LPS-induced apoptosis. MnSOD, however, was not induced in an NG-monomethyl-L-arginine (L-NMMA)-sensitive time-dependent fashion after Ad.CMV-iNOS. Other inducible genes that may be affected by NO and that may protect against potential oxidant-mediated LPS-induced apoptosis including 70-kDa heat shock protein, heme oxygenase-1, metallothionein, and Bcl-2 also were not elevated in an L-NMMA-sensitive, time-dependent fashion. Although the candidate gene product underlying NO-induced protection remains unclear, we did note that prolonged synthesis of NO inhibited LPS-induced activation of an interleukin-1beta-converting enzyme-like cysteine protease (cysteine protease protein-32-like) in a dithiothreitol-sensitive fashion, suggesting that S-nitrosylation of an important downstream target of convergence of apoptotic signals may contribute to the sensitivity of SPAECs to LPS.
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PMID:Nitric oxide inhibits lipopolysaccharide-induced apoptosis in pulmonary artery endothelial cells. 975 4

-Cytokine-induced NO production depresses myocardial contractility and has been shown to be cytotoxic to cardiac myocytes. However, the mechanisms of cytokine-induced cardiac myocyte cell death are unclear. To analyze these mechanisms in detail, we treated neonatal cardiac myocytes in serum-free culture with a combination of the macrophage-derived cytokines interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma. These cytokines caused a time-dependent induction of cardiac myocyte apoptosis, but not necrosis, beginning 72 hours after treatment, as determined by nuclear morphology, DNA internucleosomal cleavage, and cleavage of poly(ADP-ribose) polymerase, reflecting caspase activation. Apoptosis was preceded by a >50-fold induction of inducible NO synthase mRNA and the release of large amounts (5 to 8 nmol/ microgram protein) of NO metabolites (NOx) into the medium. Cell death was completely blocked by an NO synthase inhibitor and attenuated by antioxidants (N-acetylcysteine and DTT) and the caspase inhibitor ZVAD-fmk. Cytokines also mediated an NO-dependent, sustained increase in myocyte expression of the Bcl-2 homologs Bak and Bcl-x(L). The NO donor S-nitrosoglutathione also induced apoptosis and cell levels of Bak, but not of Bcl-x(L). All effects of cytokines, including poly(ADP-ribose) polymerase cleavage, could be attributed to interleukin-1beta; interferon-gamma and tumor necrosis factor-alpha had no independent effects on apoptosis or on NOx production. We conclude that cytokine toxicity to neonatal cardiac myocytes results from the induction of NO and subsequent activation of apoptosis, at least in part through the generation of oxygen free radicals. The rate and extent of this apoptosis is modulated by alterations in the cellular balance of Bak and Bcl-x(L), which respond differentially to cytokine-induced and exogenous NO and by the availability of oxidant species.
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PMID:Modulation of cytokine-induced cardiac myocyte apoptosis by nitric oxide, Bak, and Bcl-x. 991 71

The proteins Bcl-2 and Bcl-X(L) prevent apoptosis, but their mechanism of action is unclear. We examined the role of Bcl-2 and Bcl-X(L) in the regulation of cytosolic Ca(2+), nitric oxide production (NO), c-Jun NH(2)-terminal kinase (JNK) activation, and apoptosis in Jurkat T cells. Thapsigargin (TG), an inhibitor of the endoplasmic reticulum-associated Ca(2+) ATPase, was used to disrupt Ca(2+) homeostasis. TG acutely elevated intracellular free Ca(2+) and mitochondrial Ca(2+) levels and induced NO production and apoptosis in Jurkat cells transfected with vector (JT/Neo). Buffering of this Ca(2+) response with 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) or inhibiting NO synthase activity with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME) blocked TG-induced NO production and apoptosis in JT/Neo cells. By contrast, while TG produced comparable early changes in the Ca(2+) level (i.e., within 3 h) in Jurkat cells overexpressing Bcl-2 and Bcl-X(L) (JT/Bcl-2 or JT/Bcl-X(L)), NO production, late (36-h) Ca(2+) accumulation, and apoptosis were dramatically reduced compared to those in JT/Neo cells. Exposure of JT/Bcl-2 and JT/Bcl-X(L) cells to the NO donor, S-nitroso-N-acetylpenacillamine (SNAP) resulted in apoptosis comparable to that seen in JT/Neo cells. TG also activated the JNK pathway, which was blocked by L-NAME. Transient expression of a dominant negative mutant SEK1 (Lys-->Arg), an upstream kinase of JNK, prevented both TG-induced JNK activation and apoptosis. A dominant negative c-Jun mutant also reduced TG-induced apoptosis. Overexpression of Bcl-2 or Bcl-X(L) inhibited TG-induced loss in mitochondrial membrane potential, release of cytochrome c, and activation of caspase-3 and JNK. Inhibition of caspase-3 activation blocked TG-induced JNK activation, suggesting that JNK activation occurred downstream of caspase-3. Thus, TG-induced Ca(2+) release leads to NO generation followed by mitochondrial changes including cytochrome c release and caspase-3 activation. Caspase-3 activation leads to activation of the JNK pathway and apoptosis. In summary, Ca(2+)-dependent activation of NO production mediates apoptosis after TG exposure in JT/Neo cells. JT/Bcl-2 and JT/Bcl-X(L) cells are susceptible to NO-mediated apoptosis, but Bcl-2 and Bcl-X(L) protect the cells against TG-induced apoptosis by negatively regulating Ca(2+)-sensitive NO synthase activity or expression.
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PMID:Bcl-2 and Bcl-X(L) block thapsigargin-induced nitric oxide generation, c-Jun NH(2)-terminal kinase activity, and apoptosis. 1040 55

Nitric oxide (NO) is synthesized by members of the NO synthase (NOS) family. Recently the existence of a mitochondrial NOS (mtNOS), its Ca(2+) dependence, and its relevance for mitochondrial bioenergetics was reported (Ghafourifar, P., and Richter, C. (1997) FEBS Lett. 418, 291-296; Giulivi, C., Poderoso, J. J., and Boveris, A. (1998) J. Biol. Chem. 273, 11038-11043). Here we report on the possible involvement of mtNOS in apoptosis. We show that uptake of Ca(2+) by mitochondria triggers mtNOS activity and causes the release of cytochrome c from isolated mitochondria in a Bcl-2-sensitive manner. mtNOS-induced cytochrome c release was paralleled by increased lipid peroxidation. The release of cytochrome c as well as increase in lipid peroxidation were prevented by NOS inhibitors, a superoxide dismutase mimic, and a peroxynitrite scavenger. We show that mtNOS-induced cytochrome c release is not mediated via the mitochondrial permeability transition pore because the release was aggravated by cyclosporin A and abolished by blockade of mitochondrial calcium uptake by ruthenium red. We conclude that, upon Ca(2+)-induced mtNOS activation, peroxynitrite is formed within mitochondria, which causes the release of cytochrome c from isolated mitochondria, and we propose a mechanism by which elevated Ca(2+) levels induce apoptosis.
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PMID:Mitochondrial nitric-oxide synthase stimulation causes cytochrome c release from isolated mitochondria. Evidence for intramitochondrial peroxynitrite formation. 1053 11

Increased production of nitric oxide (NO) after induction of the cytokine-inducible isoform of nitric oxide synthase (iNOS or NOS2) in cardiac myocytes and other parenchymal cells within the heart may in addition to contributing to myocyte contractile dysfunction also contribute to the induction of programmed cell death (apoptosis). To investigate the mechanism(s) by which increased NO production leads to apoptosis, we examined the role of NO in primary cultures of neonatal rat ventricular myocytes (NRVMs) after induction by the cytokines interleukin-1beta (IL-1beta) and interferon gamma (IFNgamma) or exposure to the exogenous NO donor S-nitroso-N-acetylcysteine (SNAC) or peroxynitrite (ONOO(-)). Both SNAC (1 mmol/L) and ONOO(-) (100 micromol/L), but not their respective controls (ie, N-acetylcysteine and pH-inactivated ONOO(-)), induced apoptosis in confluent, serum-starved NRVMs at 48 hours. Similarly, incubation of NRVMs with IL-1beta and IFNgamma for 48 hours resulted in an increase in iNOS expression, nitrite production, and programmed cell death. Both the cytokine-induced nitrite accumulation and myocyte apoptosis could be completely prevented by the nonselective NOS inhibitor L-nitroarginine (3 mmol/L) or the specific iNOS inhibitor 2-amino-5, 6-dihydro-6-methyl-4H-1,3-thiazine (AMT, 100 micromol/L). NO-mediated myocyte apoptosis was not attenuated by the inhibition of soluble guanylyl cyclase with ODQ, nor could apoptosis be induced by the incubation of NRVMs with 1 mmol/L 8-bromo-cGMP, a cell-permeant cGMP analogue. However, NO-mediated apoptosis was significantly attenuated by the superoxide dismutase mimetic and ONOO(-) scavenger Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP, 100 micromol/L). NO/ONOO(-)-mediated apoptosis was associated with increased expression of Bax with no change in Bcl-2 mRNA abundance. Furthermore, apoptotic cell death was also confirmed in adult rat ventricular myocytes (ARVMs) when grown in heteroculture with IL-1beta- and IFNgamma-treated rat cardiac microvascular endothelial cells. Therefore, cytokine-induced apoptosis in NRVMs and ARVMs is mediated by iNOS induction, ONOO(-), and associated with an increase in Bax levels.
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PMID:Cytokine-mediated apoptosis in cardiac myocytes: the role of inducible nitric oxide synthase induction and peroxynitrite generation. 1053 56

This study addresses the effects of IL-1 beta on apoptosis induced by agonistic anti-CD95 (Fas) Ab. IL-1 beta inhibited anti-CD95 Ab-induced apoptosis in all preparations of normal human articular chondrocytes tested. Inhibitors of nitric oxide synthase or cyclooxygenase did not influence the protective effect of IL-1 beta, indicating that nitric oxide and PGs were not involved in the modulation of CD95-induced apoptosis. However, when the IL-1 beta-dependent induction of NF-kappa B was inhibited, the antiapoptotic effect of IL-1 beta was partially reversed, suggesting that NF-kappa B-mediated gene activation is part of the protective mechanism. In addition, IL-1 beta significantly increased the expression of Bcl-2. The protein tyrosine kinase inhibitor herbimycin A completely eliminated the protective effect of IL-1 beta on CD95-induced apoptosis. These findings suggest that IL-1 beta modulates the CD95 death cascade in chondrocytes by mechanisms that involve tyrosine phosphorylation events and NF-kappa B-dependent gene activation.
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PMID:IL-1 beta protects human chondrocytes from CD95-induced apoptosis. 1065 79

Adrenomedullin, which was discovered as a vasodilating peptide, has been reported to be produced in various organs, in which adrenomedullin regulates not only vascular tone but also cell proliferation and differentiation in an autocrine/paracrine manner. We evaluated the effect of adrenomedullin on endothelial cell apoptosis. Human umbilical vein endothelial cells underwent apoptosis when cultured in serum-free medium. Treatment with adrenomedullin reduced the number of cells with pyknotic nuclei (Hoechst 33258 staining) and inhibited cell death (dimethylthiazol-diphenyltetrazolium bromide assay) in a dose-dependent manner. The administration of adrenomedullin did not alter the expression levels of Bcl-2 family proteins. Experiments with analogs of cAMP or a cAMP-elevating agonist demonstrated that elevation of the intracellular cAMP concentration does not mediate the antiapoptotic effect of adrenomedullin. The coadministration of N-nitro-L-arginine methyl ester (2 mmol/L), an inhibitor of nitric oxide synthase, abrogated the effect of adrenomedullin. Lower doses of sodium nitroprusside (1 to 10 micromol/L), a nitric oxide donor, mimicked the antiapoptotic effect of adrenomedullin. The antiapoptotic effect of sodium nitroprusside was not attenuated by the inhibition of soluble guanylyl cyclase with 1 micromol/L oxadiazolo-quinoxalin-1-one nor could apoptosis be inhibited by the incubation of human umbilical vein endothelial cells with 1 mmol/L 8-bromo-cGMP, a cell-permeant cGMP analog. These results indicate that adrenomedullin and nitric oxide inhibit endothelial cell apoptosis via a cGMP-independent mechanism.
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PMID:Adrenomedullin and nitric oxide inhibit human endothelial cell apoptosis via a cyclic GMP-independent mechanism. 1090 17

The present study aimed to investigate the role of nitric oxide (NO) in regression of the human corpus luteum. We therefore examined the effect of both NO and human chorionic gonadotrophin (HCG) on luteal cell apoptosis, and Bcl-2 production. The effect of NO on oestrogen production during corpus luteum regression was also studied. Slices from corpus luteum collected throughout the luteal phase were incubated for 4 h with the nitric oxide synthase (NOS) substrate, L-arginine (L-Arg, 1 mmol/l), the NOS inhibitor N-monomethyl-L-arginine (L-NMMA) (1 mmol/l), or with HCG (10 IU/ml). Oestradiol concentrations were determined by radioimmunoassay; Bcl-2 concentrations were measured by enzyme-linked immunosorbent assay; apoptosis was detected in-situ by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling; and inducible nitric oxide synthase (iNOS) was assessed by immunohistochemistry. Consistent with our previous findings, L-Arg elicited an inhibitory action on the production of oestradiol (P< 0.05). The number of apoptotic cells increased (P<0.05) from early to late corpus luteum, as did the number of cells positive for the expression of iNOS. The percentage of apoptotic cells in mid and late luteal phase was increased by L-Arg (56% and 310% respectively; P <0.05), and decreased by L-NMMA and HCG. Although no changes were observed in Bcl-2 concentration during the corpus luteum life span, L-Arg inhibited, and HCG augmented, Bcl-2 production (P<0.05) from mid and late corpus luteum cells in vitro. In summary, these results suggest that the opposite actions of L-Arg and HCG on human corpus luteum viability may, in part, be mediated by changes in the level of the anti-apoptotic activities caused by oestradiol and Bcl-2 protein.
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PMID:Nitric oxide induces apoptosis in the human corpus luteum in vitro. 1090 76

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